By Guzelmeric, Kadir Alkan, Nevriye; Pirimoglu, Meltem; Unal, Orhan; Turan, Cem
Abstract Background. Women with polycystic ovary syndrome (PCOS) are insulin-resistant and have increased risk for type 2 diabetes mellitus (T2DM) and coronary heart disease (CHD). But it is controversial whether the increased risk of CHD and T2DM is associated with endocrine abnormalities occurring as a consequence of PCOS or whether it is related to obesity or metabolic changes frequently seen in women with PCOS.
Objective. Since both homocysteine (Hey) and C-reactive protein (CRP) are supposed to predict T2DM and CHD, we investigated their possible relationship with insulin resistance, obesity, hyperandrogenemia and metabolic alterations in 44 PCOS women and 26 healthy controls matched by age and body mass index (BMI).
Results. Hey and CRP levels were significantly elevated in PCOS women compared with controls (13.30 +- 4.81 vs. 9.02 +- 3.36 [mu]mol/ l, p 0.05). Multiple regression analysis revealed BMI as the major factor examined that influenced both Hey and CRP levels.
Conclusions. In PCOS women, plasma levels of Hey and CRP were significantly elevated compared with age- and BMI-matched controls. Although most of the PCOS-related endocrine and metabolic changes are related to elevated plasma Hey and CRP levels in PCOS women, BMI seems to be the major factor determining CHD and T2DM in women with PCOS.
Keywords: Homocysteine, C-reactive protein, insulin resistance, body mass index, polycystic ovary syndrome
Introduction
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of women of reproductive age and is associated with long- term health risks, including type 2 diabetes mellitus (T2DM) and coronary artery disease [1,2]. In particular, the insulin resistance, hyperandrogenemia and dyslipidemia associated with PCOS are major risk factors for coronary heart disease (CHD) [1-3].
Hyperhomocysteinemia is another important risk factor for the development of coronary and thromboembolic diseases [4]. Homocysteine (Hey) is a sulfur-containing amino acid formed during the metabolism of methionine [5]. Classic hyperhomocysteinemia has been characterized as the accumulation of Hey due to defects in enzymatic pathways [4]. Elevated plasma Hey induces oxidation of low- density lipoprotein (LDL), proliferation of smooth muscle cells, increased platelet adhesiveness and endothelial cytotoxicity [4-6]. It has been reported that insulin levels are associated with increased plasma levels of Hcy in healthy, non-obese subjects [7]. In addition, an association between insulin resistance and plasma Hcy concentrations has been described [8].
Inflammation is thought to play a key role in the pathophysiological mechanism of atherosclerosis and CHD [9]. C- reactive protein (CRP) is known to be a significant predictor of CHD and future cardiovascular events [9,10]. Recently, low-grade chronic inflammation has been linked to obesity and the insulin resistance syndromes, such as PCOS [11].
In the present study we investigated the possible relationships of plasma Hey and CRP levels with insulin resistance, obesity and known biochemical markers in PCOS women.
Materials and methods
Subjects
Between December 2004 and July 2005, 44 women (mean age: 23.5 + 4.7 years) with PCOS were enrolled in this study. The diagnosis of PCOS was made using menstrual, laboratory and ultrasound criteria. Menstrual criteria included oligo- and amenorrhea (cycle length irregular, >45 days or 2) and elevated plasma testosterone levels at the upper limit of the range (0.10-1.30 ng/ml). Ultrasound criteria used for diagnosis were normal or enlarged ovaries with the presence of microcysts (2-8 mm in diameter) and a hyperechogenic stroma. Serum prolactin, thyroid hormone and 17-hydroxyprogesterone (17- OHP) determinations were done on all patients and were within normal limits. All patients included in the study had not taken ovulation induction agents, glucocorticosteroids, antiandrogens or antihypertensive medications in the previous 6 months.
Twenty-six non-hirsute, regular cycling women, matched to cases in terms of body mass index (BMI) and age (mean age: 25.85 +- 5.82 years), who had been subject to the same menstrual, laboratory and ultrasound tests and had no ultrasound or clinical signs of PCOS, served as controls. Smoking status, coffee consumption and lifestyle were similar between cases and controls.
Each patient was examined to evaluate hirsutism according to the modified Ferriman-Gallwey score. BMI was computed as weight divided by the square of height (kg/m^sup 2^).
Laboratory tests
Hormonal assays and ultrasonography were performed during the early follicular phase, between the 3rd and the 5th days of the patients’ spontaneous or progestin-induced menstrual cycle. Blood samples were taken early in the morning subsequent to an overnight fast. Plasma was removed and stored at -20[degrees]C until analyzed.
Blood glucose was measured by the glucose dehydrogenase method on the Gobas Mira laboratory system (Roche Diagnostics GmbH, Mannheim, Germany). Insulin was measured by a commercial radioimmunoassay kit (Pharmacia, Stockholm, Sweden) with a lower limit of sensitivity of
Total plasma Hcy was assayed by fluorescence polarization immunoassay by the IMX System (Abbott Diagnostics, Wiesbaden, Germany). The intra-assay CV was 2.8% and inter-assay CV was 3.8- 5.0%.
Insulin resistance was determined from fasting glucose and insulin as HOMA-IR (homeostasis model assessment-insulin resistance) index: HOMA-IR= [glucose (mmol/1) x insulin (mIU/l)]/22.5. Correlations between clinical or biochemical parameters and insulin resistance were calculated using this index.
CRP was measured with a turbidimetric test (Wako, Neus, Germany) with the Gobas Mira laboratory system. Again all samples were analyzed in one assay. The intra- and inter-assay CVs were 17% and 12%, respectively.
Total testosterone was assayed by automated electrochemiluminescence immunoassay (Roche), while radioimmunoassays were used for free testosterone, 17-OHP and dehydroepiandrosterone sulfate (DHEAS) (Diagnostic Products Corporation, Los Angles, CA, USA). The intra- or inter-assay CVs were 9.5% and 11.8% for 17-OHP; 4.9% and 7.5% for total testosterone; 6.2% and 9.7% for free testosterone; and 5.2% and 7.7% for DHEAS.
Statistical analyses
All results are expressed as mean +- standard deviation, unless otherwise stated. Differences between the two groups were evaluated with use of the Student t test and the non-parametric Mann-Whitney U test for variables with persisting skewed distribution. The Kolmogorov-Smirnov test was used to evaluate whether the variables were normally distributed or not. Pearson’s correlation test was employed to analyze the correlations between parameters. In addition, the significance of the correlation and the relative contribution of each variable were calculated by single or multiple regression analyses, respectively. Significance was set as p
Results
The characteristics of the PCOS patients are summarized in Table I. As anticipated, in the PCOS women, total testosterone, free testosterone, LH/ FSH ratio, 17-OHP and DHEAS were significantly increased (p
The groups were similar in age. Their risk factors related to cardiovascular disease were significantly different, although BMI was not significantly different in PCOS cases compared with controls (26.50 +- 4.7 vs. 24.13 +- 3.59 kg/m^sup 2^, p > 0.05). Ferriman- Gallwey scores were significantly higher in the PCOS patients than in the control group (13.95 +- 6.7 vs. 5.27 +- 1.5, p
Table I. Clinical, hormonal and metabolic features of women with polycystic ovary syndrome (PCOS) and healthy controls.
Pearson correlations were calculated in order to define parameters associated with plasma levels of Hcy and CRP. There was no correlation between Hcy and CRP (r=0.171, p=0.05) as two risk markers. While plasma Hcy levels were correlated with BMI, LH/FSH ratio, total testosterone, free testosterone, triglyceride, insulin levels and HOMA-IR, CRP was correlated with BMI, total cholesterol, triglyceride, LJDL cholesterol, insulin levels and HOMA-IR (Tables II and III). There was no correlation of CRP with parameters of PCOS such as total testosterone and LH/FSH ratio. Plasma Hcy levels were associated with obesity, metabolic alterations or endocrine changes in PCOS women. Linear regression models were calculated to further investigate whether obesity (BMI) and insulin resistance (HOMA-IR) are predictors of Hcy or CRP concentrations. HOMA-IR was not associated with Hcy and CRP levels, after inclusion of BMI into the model. Both Hcy and CRP concentrations were significantly affected by BMI with adjusted R^sup 2^ of 0.12 and 0.14, respectively (Figure 2). In the case of Hcy, the beta coefficient of the model was 0.349 (p = 0.003) for BMI and 0.182 (p = 0.13) for HOMA-IR. With CRP as the dependent variable, the beta coefficient for BMI was 0.383 (p = 0.001) and for HOMA-IR was 0.179 (p = 0.131). In addition, PCOS status was not a significant independent factor affecting Hcy and CRP levels. Obesity seems to be the dominant factor determining Hcy and CRP values in PCOS women. Finally, multiple linear regression analysis showed that hormone levels and metabolic alterations are not predictors of CRP or Hcy levels in both PCOS and control women.
Figure 1. Serum homocysteine (Hcy) and C-reactive protein (CRP) concentrations in 44 patients with polycystic ovary syndrome (PCOS) and 26 age-matched healthy controls. Results are expressed as means, with standard deviation shown by vertical bars. For discussion, see text.
Discussion
Moderately increased total Hcy level is associated with an increased risk of atherosclerosis [6,7]. High Hcy concentrations may be associated with demographic, genetic, nutritional or metabolic factors [12-15]. Hyperhomocysteinemia induces sustained injury to the arterial endothelial cell, which accelerates the development of thrombosis and atherosclerosis [4,6,16]. On the other hand, the results of recent studies suggested that CRP, rather than being only a marker of low-grade inflammation, may directly promote endothelial dysfunction and complement activation and therefore could play an active role in atherogenesis [17,18]. Insulin resistance syndromes such as T2DM and PCOS have been linked to elevated plasma Hey levels [11,19]. Moreover, it has been shown that women with PCOS, as in T2DM, have significantly increased CRP concentrations relative to healthy subjects [10,20].
Table II. Correlations between homocysteine (Hcy) and the investigated variables.
Table III. Correlation between C-reactive protein (CRP) and the investigated variables.
Figure 2. Plasma homocysteine (Hcy) and C-reactive protein (CRP) as a function of body mass index (BMI).
In the present study both plasma Hcy levels and CRP concentrations in PCOS women were significantly elevated, with values higher than the upper range limits, compared with healthy controls. In comparison to matched controls PCOS women had greater serum insulin concentrations and HOMA-IR, confirming previous data.
While plasma Hcy levels were correlated with BMI, LH/FSH ratio, total testosterone, free testosterone, triglyceride, insulin levels and HOMA-IR, CRP levels were correlated with BMI, total cholesterol, triglycerides, LDL cholesterol, insulin levels and HOMA-IR. There was no correlation of CRP with parameters of PCOS such as total testosterone and LH/FSH ratio. This indicates that obesity and metabolic alterations associated with PCOS have more impact than other markers on chronic inflammation in PCOS women. This observation is consistent with previous data demonstrating a lack of association between total testosterone and lipid and lipoprotein subfraction concentrations in women with PCOS [21]. In another recent study, urinary sex hormone metabolites were not related to an increased CHD risk in women [22]. Plasma Hcy levels were associated with obesity, metabolic alterations or endocrine changes in PCOS women. Hcy and CRP were not dependent on one another although both were elevated in PCOS patients in comparison with BMI-matched controls.
Multiple regression analysis demonstrated that hormone levels and impaired lipid profile were not predictors of elevated levels of Hcy or CRP in women with PCOS, suggesting that hyperhomocysteinemia or high CRP concentrations are not affected by hyperandrogenemia or hyperlipidemia. In our study, although both high Hcy and CRP levels correlated with BMI and insulin sensitivity, stepwise linear regression analysis showed that BMI was the only strong predictor of serum Hcy and CRP concentrations both in PCOS patients and controls. In contrast, most studies have demonstrated insulin resistance as the determinant of Hcy in PCOS women and not necessarily body weight [8,23]. But there are studies showing the significance of the relationship between insulin levels and hyperhomocysteinemia as debatable. Giltay and colleagues found a significant association between high insulin levels and elevated Hcy plasma levels in healthy, nonobese subjects [7]. On the other hand, Abbasi and associates showed that plasma Hcy levels did not vary as a function of insulin-mediated glucose disposal [24]. Kilic-Okman and co- workers showed that age, BMI and insulin resistance were not predictors of Hcy [25], while Schachter’s group found that insulin resistance and hyperinsulinemia in patients with PCOS was associated with elevated plasma Hcy, regardless of body weight [23].
A number of variables that were not examined in this study, such as renal function, vitamin B status and enzyme dysfunction states, influence Hcy levels. Plasma Hcy levels may be influenced by genetic and non-genetic factors (diet, age, pregnancy, menstrual cycle) [12- 14]. In our study, the patient group and the control group were in similar age and all blood samples were collected during menstruation. Because of the conflicting data on the relationship between insulin and hyperhomocysteinemia in PCOS, we also included CRP concentrations as another cardiovascular risk parameter.
It has recently been reported that visceral adiposity correlates with CRP concentrations independently of total adiposity in a cross- sectional study of men [27]. This shows that adipose tissue-derived cytokine expression (tumor necrosis factor-alpha and interleukin-6) may be an important contributor to low-grade chronic inflammation. That means that accumulation of visceral adipose tissue may be the underlying feature of the metabolic syndrome and of low-grade chronic inflammation. These observations would explain the correlation of insulin resistance to CRP, as declined by Festa and colleagues [26].
Although not measured in our study, women with PCOS are likely to have increased visceral fat mass relative to BMI-matched controls. Mohlig and collaborators also revealed that BMI rather than HOMA-IR index was the dominant parameter determining serum interluekin-6 and CRP concentrations in women with PCOS [28].
In conclusion, we have shown that CRP concentrations, a marker of low-grade inflammation and CHD risk, measured using a highly sensitive assay, are significantly increased in PCOS women with elevated Hcy levels relative to those in BMI-matched healthy women. The main factor predicting Hcy and CRP levels in PCOS was BMI. Thus CHD or T2DM risk level in PCOS appears to be enhanced mainly by obesity, regardless of endocrine abnormalities. Further prospective studies are needed in order to confirm whether raised Hcy and CRP levels in young PCOS patients serve as a real risk marker depending on BMI.
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This paper was first published online on iFirst on 6 August 2007
KADIR GUZELMERIC, NEVRIYE ALKAN5 MELTEM PIRIMOGLU, ORHAN UNAL, &
CEM TURAN
Department of Obstetrics and Gynecology, Dr Lutfi Kirdar Kanal Education and Research Hospital, Istanbul, Turkey
(Received 8 February 2007; revised 4 April 2007; accepted 4 July 2007)
Correspondence: K. Guzelmeric, 34 Ada Yesim Sitesi Blok 12 Daire 8 Atasehir, 34758 Kadikoy, Istanbul, Turkey. Tel: 90 216 4566146. Fax: 90 216 3064294. E-mail: [email protected]
Copyright Taylor & Francis Ltd. Sep 2007
(c) 2007 Gynecological Endocrinology. Provided by ProQuest Information and Learning. All rights Reserved.
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